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5% agarose gel stained by ethidium bromide for and virus, respectively. Also, a dual-band at 148 and 167 was observed in multiplex PCR. Results of real-time PCR showed a limit of detection about 0.1 of plasmid/ . In conclusion, the designed construct can be used as a positive control for an accurate diagnosis of these micro-organisms without any biological danger for laboratory staff. So, this method is useful for diagnosis of these agents in food, water, and blood samples. In conclusion, the designed construct